The present invention relates to an immortal cell line (GF-1) derived from the fin tissue of grouper Epinephelus coioides and the method of establishing the GF-1 cell line. The GF-1 cell line is susceptible to a number of aquatic viruses, including, but not limited to, Infectious Pancreatic Necrosis Virus (IPNV), Eel Herpes Virus Formosa (EHVF), and Nervous Necrosis Virus (NNV). This invention also relates to the method of mass producing and purifying the aquatic viruses using an immortal cell line from grouper such as the GF-1 cell line as a host. Additionally, this invention relates to an anti-NNV antibody and the method of producing the anti-NNV antibody. Finally, this invention relates to a vaccine of NNV and the method for protecting fish against NNV infection.
Nervous necrosis virus (NNV), a pathogen found in many varieties of hatchery-reared marine fish, has caused mass mortality of such fish at their larval or juvenile stages. NNV belongs to the family Nodaviridae. Fish nodaviruses isolated from different species (such as SJNNV, BFNNV, JFNNV, TPNNV, RGNNV, GNNV etc.) are closely related to each other owing to the high similarity of the conserved region of their coat protein genes. NNV, also named as fish encephalitis virus (FEV) and piscine neuropathy nodavirus (PNN), is an unenveloped spherical virus with particles sized between 25 and 34 nm. The virus is characterized by vacuolation of the nerve tissues. Viral Nervous Necrosis (VNN) disease has been found in many countries under various names such as viral fish encephalitis, fish encephalomyelitis, cardiac myopathy syndrome. The hosts of NNV include many species of marine fish, for example: parrotfish, sea bass, turbot, grouper, stripped jack, tiger puffer, berfin flounder, halibut, barramundi, and spotted wolffish.
According to the statistics shown in 1993, approximately 159 fish cell lines have been established which have demonstrated a capacity for growing fish viruses (Fryer and Lannan, J. Tissue Culture Method (1994), 10:57-94). Most of these cell lines are derived from the tissues of freshwater fish. There are only thirty-four cell lines which are originated from marine fish. Although some of the fish cell lines, which include RTG-2, CHSE-214, BF2, SBL, FHM, EPC, have been tested for the susceptibility of fish nodavirus, none of these cells lines has shown cytopathic effects (CPE) after viral inoculations.
In 1996, SSN-1 cell line, a cell line derived from striped snakehead Channa Striatus, has been successfully used for isolating sea bass nodavirus (Frerichs et al., J. General Virology (1996)77:20672071). However, SSN-1 cell line has been known to be persistently contaminated with C-type retrovirus (Frerichs et al., J. General Virology (1991) 72:2537-2539). Therefore, it is not suitable for the production of fish nodavirus.
Viral diseases cannot be cured by therapeutic reagents. The best ways to contain viral diseases include prevention through early detection and the development of vaccines. In either way, the understanding of the biological, biochemical, and serological characteristics of the virus is fundamentally required, which in turn relies on the industry to have the capacity of mass producing the pure form of viruses, preferably through an in vitro cell culture system. Therefore, the development of a new cell line which can be susceptible to fish nodavirus is desperately in demand in order to control the wide spread of fish viral diseases due to fish nodavirus infection.
Grouper is an important hatchery fish in Taiwan. In recent years, there have been several reports regarding the establishment of cell lines derived from grouper. For example, Chen et. al. (Japan Scientific Society Press (Tokyo) (1988) 218-227) have reported their establishment of several cell lines from the fin and kidney tissues of grouper Epinephelus awoara. Lee (Master Thesis from the Department of Zoology at the National Taiwan University, 1993) also has reported his establishment of the cell lines derived from the eye pigment cells and brain tissue of grouper Epinephelus amblycephalus. However, Chen et al. do not provide sufficient data in support of the claim for immortality in their cell lines and Lee expressly indicates in his thesis that his grouper cell lines are not immortal. Moreover, neither Chen et al.""s nor Lee""s cell lines are susceptible to fish nodavirus.
Recently, severe mortality among groupers has repeatedly occurred which is caused primarily by nodavirus. As present, fish nodavirus has been discovered in grouper and can be isolated from moribund grouper which possess symptoms of VNN disease (Chi et al., J. Fish Disease (1997) 20:185-193). Electron microscopic examination of the tissues from grouper shows that, in addition to nodavirus infection, grouper is susceptible to other viral infections (Chi, COA Fisheries Series No.61, Reports on Fish Disease Research (1997) 18:59-69). The fact that some viruses have host specificity makes a cell line derived from grouper more appropriate for investigating the specific viruses isolated from grouper.
In the invention to be presented below, an immortal cell line derived from the fin tissue of grouper Epinephelus coioides (Hamilton) will be introduced: The cell line of the present invention is susceptible to various viruses, particularly fish nodavirus such as GNNV. Using the present cell line, various aquatic viruses can be mass produced and purified. The purified viruses are useful for antibody and vaccine production to protect fish from viral infections.
A first embodiment of the present invention provides for an immortal cell line derived from grouper, preferably, an immortal cell line (GF-1) which is derived from the fin tissue of grouper Epinephelus coioides. GF-1 is susceptible to, and can mass produce viruses which include, but are not limited to, viruses from the families of Birnaviridae (such as infectious pancreatic necrosis virus [IPNV]), Herpesviridae (such as eel herpes virus Formosa [EHVF]), Reoviridae (such as hard clam reovirus [HCRV]), and Nodaviridae (such as grouper nervous necrosis virus [GNNV]).
The first embodiment also provides for a method of establishing an immortal cell line. The method comprises the steps of : (1) establishing a primary cell culture by placing cells released from the fin tissue of grouper Epinephelus coioides in a tissue culture flask to form a monolayer of cells; (2) subculturing and maintaining the monolayer of cells in a media suitable for cell subculturing; and (3) monitoring a transformation of cells which is characterized by a change in chromosome number distribution, plating efficiency, fetal bovine serum (FBS) requirement , and susceptibility to aquatic viruses, particularly fish nodavirus such as GNNV.
A second embodiment of the invention provides for a method for growing a virus using the immortal cell line derived from grouper, preferably the GF-1 cell line. The method comprises the steps of. (1) inoculating the virus into the cell line; and (2) incubating the cell line in a nutrient medium suitable for growth and replication of the virus. The viruses which are susceptible to and can be replicated in the immortal cell line include viruses from the families of Birnaviridae, Herpesviridae, Reoviridae, and Nodaviridae, and, in particular, lPNV of Birnaviridae, EHVF of Herpesviridae, HCRV of Reoviridae, and GNNV of Nodaviridae.
The second embodiment also provides for methods of mass producing the viruses using the immortal grouper cell line, purifying the viruses, and detecting the viruses in the cell line. The method for mass producing the virus comprises: (1) inoculating the virus into the grouper cell line; (2) incubating the cell line in a nutrient medium suitable for growth and replication of the virus; and (3) harvesting the virus from the cell line.
The method for purifying a virus comprises: (1) inoculating the virus into the grouper cell line; (2) incubating the cell line in a nutrient medium suitable for growth and replication of the virus until the appearance of cytopathic effects (CPE); (3) harvesting the virus from the cell line; and (4) purifying the virus using density gradient centrifugation. The preferable density gradient is a CsCl density gradient. However, other density gradients which yield a sufficient virus harvest are also within the scope of the invention.
The present method for detecting a virus in the immortal grouper cell line comprises: observing a development of cytopathic effects (CPE) in the cell line under microscope. The virus can be further confirmed by the electron microscopic method which comprises the steps of. (1) fixing the cell line in glutaraldehyde and osmium tetraoxide; (2) performing ultrathin sectioning of the fixed cells; and (3) detecting viral particles in the ultrathin section of the fixed cell line under an electron microscope.
There are four methods which contribute to the specific detection of NNV in the immortal grouper cell line after the CPE is detected in the cell line. A first method uses the polymerase chain reaction (PCR), which comprises the steps of: (1) extracting a viral RNA from the cell line; (2) amplifying the viral RNA by PCR using a reverse primer (SEQ ID NO. 1) and a forward primer (SEQ ID NO.2). A second method uses a western immunoblot, which comprises the steps of. (1) extracting the viral protein from the cell line; (2) electrophoresizing the viral protein in an SDS-polyacrylamide gel; and analyzing the polyacrylamide gel by western immunoblot using an anti-NNV serum. A third method uses an enzyme-linked immunosorbent assay (ELISA) method to detect NNV protein. A fourth method uses immunofluorescent staining by coupling fluorescein isothiocyanate (FITC) conjugated goat anti-mouse antibodies with mouse anti-NNV serum to thereby detect NNV within the cells.
A third embodiment of the invention provides for an anti-NNV antibody and a method of making the anti-NNV antibody. The anti-NNV antibody is preferably a monoclonal antibody. The anti-NNV antibody is prepared by administering an effective amount of NNV to a suitable animal, preferably a mouse or a rabbit, to stimulate an immunoresponse in the animal. The NNV used for making the anti-NNV antibody is preferably the one harvested from the grouper cell line and further purified by a CsCl density gradient centrifugation (NNV has a buoyant density of approximately 1.34 g/ml in CsCl).
A fourth embodiment of the invention provides for a vaccine to NNV and a method for protecting fish against NNV infection. The NNV vaccine comprises an immunogenically effective amount of killed NNV. The vaccine further comprises a material selected from the group consisting of adjuvants, plasticizers, pharmaceutical excipients, diluents, carriers, binders, lubricants, glidants and aesthetic compounds, and combinations thereof. The vaccine can be administered orally or by injection. Oral administration is the preferred method of vaccinating fish. For the orally administered vaccine, an enteric coating which is impervious to dissolution in the stomach of fish can be added to the vaccine. The NNV useful for making the anti-NNV antibody is preferably the one harvested from the grouper cell line and further purified by a CsCl density gradient centrifugation (NNV has a buoyant density of approximately 1.34 g/ml in CsCl). The NNV is preferably inactivated. The present method for protecting fish against NNV infection comprises: administrating an effective amount of the NNV vaccine to a fish.